A combination of DNA vaccines targeting human papillomavirus type 16 E6 and E7 generates potent antitumor effects

ABSTRACT Our results presented here demonstrate that the most abundant human papillomavirus type 16 (HPV-16) mRNAs expressing the viral oncogenes E6 and E7 are regulated by cellular ASF/SF2, itself defined as a proto-oncogene and overexpressed in cervical cancer cells. We show that the most frequently used 3′-splice site on the HPV-16 genome, site SA3358, which is used to produce primarily E4, E6, and E7 mRNAs, is regulated by ASF/SF2. Splice site SA3358 is immediately followed by 15 potential binding sites for the splicing factor ASF/SF2. Recombinant ASF/SF2 binds to the cluster of ASF/SF2 sites. Mutational inactivation of all 15 sites abolished splicing to SA3358 and redirected splicing to the downstream-located, late 3′-splice site SA5639. Overexpression of a mutant ASF/SF2 protein that lacks the RS domain, also totally inhibited the usage of SA3358 and redirected splicing to the late 3′-splice site SA5639. The 15 ASF/SF2 binding sites could be replaced by an ASF/SF2-dependent, HIV-1-derived splicing enhancer named GAR. This enhancer was also inhibited by the mutant ASF/SF2 protein that lacks the RS domain. Finally, silencer RNA (siRNA)-mediated knockdown of ASF/SF2 caused a reduction in spliced HPV-16 mRNA levels. Taken together, our results demonstrate that the major HPV-16 3′-splice site SA3358 is dependent on ASF/SF2. SA3358 is used by the most abundantly expressed HPV-16 mRNAs, including those encoding E6 and E7. High levels of ASF/SF2 may therefore be a requirement for progression to cervical cancer. This is supported by our earlier findings that ASF/SF2 is overexpressed in high-grade cervical lesions and cervical cancer.

Download Full-text

Genetic stability of a recombinant adenovirus vaccine vector seed library expressing human papillomavirus type 16 E6 and E7 proteins

Experimental and Therapeutic Medicine ◽

10.3892/etm.2015.2268 ◽

2015 ◽

Vol 9 (4) ◽

pp. 1161-1165 ◽

Cited By ~ 1

Author(s):

JIE WU ◽

KE-DA CHEN ◽

MENG GAO ◽

GANG CHEN ◽

SU-FENG JIN ◽

...

Keyword(s):

Human Papillomavirus ◽

Genetic Stability ◽

Recombinant Adenovirus ◽

Vaccine Vector ◽

Human Papillomavirus Type 16 ◽

Adenovirus Vaccine ◽

E6 And E7 ◽

E7 Proteins

Download Full-text

Adeno-Associated Virus Major Rep78 Protein Disrupts Binding of TATA-Binding Protein to the p97 Promoter of Human Papillomavirus Type 16

Journal of Virology ◽

10.1128/jvi.74.5.2459-2465.2000 ◽

2000 ◽

Vol 74 (5) ◽

pp. 2459-2465 ◽

Cited By ~ 17

Author(s):

Pei-Fen Su ◽

Shu-Yuan Chiang ◽

Cheng-Wen Wu ◽

Felicia Y.-H. Wu

Keyword(s):

Human Papillomavirus ◽

Binding Protein ◽

Core Promoter ◽

Tata Binding Protein ◽

Tata Box ◽

Dependent Manner ◽

Hpv 16 ◽

Adeno Associated Virus ◽

Human Papillomavirus Type 16 ◽

E6 And E7

ABSTRACT Adeno-associated virus type 2 (AAV) is known to inhibit the promoter activities of several oncogenes and viral genes, including the human papillomavirus type 16 (HPV-16) E6 and E7 transforming genes. However, the target elements of AAV on the long control region (LCR) upstream of E6 and E7 oncogenes are elusive. A chloramphenicol acetyltransferase assay was performed to study the effect of AAV on the transcription activity of the HPV-16 LCR in SiHa (HPV-positive) and C-33A (HPV-negative) cells. The results reveal that (i) AAV inhibited HPV-16 LCR activity in a dose-dependent manner, (ii) AAV-mediated inhibition did not require the HPV gene products, and (iii) the AAV replication gene product Rep78 was involved in the inhibition. Deletion mutation analyses of the HPV-16 LCR showed that regulatory elements outside the core promoter region of the LCR may not be direct targets of AAV-mediated inhibition. Further study with the electrophoretic mobility shift assay demonstrated that Rep78 interfered with the binding of TATA-binding protein (TBP) to the TATA box of the p97 core promoter more significantly than it disrupted the preformed TBP-TATA complex. These data thus suggest that Rep78 may inhibit transcription initiation of the HPV-16 LCR by disrupting the interaction between TBP and the TATA box of the p97 core promoter.

Download Full-text

Restoration of telomeres in human papillomavirus-immortalized human anogenital epithelial cells

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.961-969.1994 ◽

1994 ◽

Vol 14 (2) ◽

pp. 961-969

Author(s):

A J Klingelhutz ◽

S A Barber ◽

P P Smith ◽

K Dyer ◽

J K McDougall

Keyword(s):

Human Papillomavirus ◽

Epithelial Cells ◽

Telomere Length ◽

Open Reading Frames ◽

Population Doubling ◽

Telomere Shortening ◽

Human Papillomavirus Type 16 ◽

Immortalized Cells ◽

E6 And E7

Loss of telomeres has been hypothesized to be important in cellular senescence and may play a role in carcinogenesis. In this study, we have measured telomere length in association with the immortalization and transformation of human cervical and foreskin epithelial cells by the human papillomavirus type 16 or 18 E6 and E7 open reading frames. By using a telomeric TTAGGG repeat probe, it was shown that the telomeres of precrisis normal and E6-, E7-, and E6/E7-expressing cells gradually shortened with passaging (30 to 100 bp per population doubling). Cells that expressed both E6 and E7 went through a crisis period and gave rise to immortalized lines. In contrast to precrisis cells, E6/E7-immortalized cells generally showed an increase in telomere length as they were passaged in culture, with some later passage lines having telomeres that were similar to or longer than the earliest-passage precrisis cells examined. No consistent association could be made between telomere length and tumorigenicity of cells in nude mice. However, of the three cell lines that grew in vivo, two had long telomeres, thus arguing against the hypothesis that cancer cells favor shortened telomeres. Our results indicate that arrest of telomere shortening may be important in human papillomavirus-associated immortalization and that restoration of telomere length may be advantageous to cells with regard to their ability to proliferate.

Download Full-text

Immune Responses and Therapeutic Antitumor Effects of an Experimental DNA Vaccine Encoding Human Papillomavirus Type 16 Oncoproteins Genetically Fused to Herpesvirus Glycoprotein D

Clinical and Vaccine Immunology ◽

10.1128/cvi.00264-10 ◽

2010 ◽

Vol 17 (10) ◽

pp. 1576-1583 ◽

Cited By ~ 17

Author(s):

Mariana O. Diniz ◽

Marcio O. Lasaro ◽

Hildegund C. Ertl ◽

Luís C. S. Ferreira

Keyword(s):

Human Papillomavirus ◽

T Cell ◽

Immune Responses ◽

Dna Vaccine ◽

T Cell Responses ◽

Glycoprotein D ◽

Antitumor Effects ◽

Hpv 16 ◽

Human Papillomavirus Type 16 ◽

Cell Responses

ABSTRACT Recombinant adenovirus or DNA vaccines encoding herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) genetically fused to human papillomavirus type 16 (HPV-16) oncoproteins (E5, E6, and E7) induce antigen-specific CD8+ T-cell responses and confer preventive resistance to transplantable murine tumor cells (TC-1 cells). In the present report, we characterized some previously uncovered aspects concerning the induction of CD8+ T-cell responses and the therapeutic anticancer effects achieved in C57BL/6 mice immunized with pgD-E7E6E5 previously challenged with TC-1 cells. Concerning the characterization of the immune responses elicited in mice vaccinated with pgD-E7E6E5, we determined the effect of the CD4+ T-cell requirement, longevity, and dose-dependent activation on the E7-specific CD8+ T-cell responses. In addition, we determined the priming/boosting properties of pgD-E7E6E5 when used in combination with a recombinant serotype 68 adenovirus (AdC68) vector encoding the same chimeric antigen. Mice challenged with TC-1 cells and then immunized with three doses of pgD-E7E6E5 elicited CD8+ T-cell responses, measured by intracellular gamma interferon (IFN-γ) and CD107a accumulation, to the three HPV-16 oncoproteins and displayed in vivo antigen-specific cytolytic activity, as demonstrated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled target cells pulsed with oligopeptides corresponding to the H-2Db -restricted immunodominant epitopes of the E7, E6, or E5 oncoprotein. Up to 70% of the mice challenged with 5 × 105 TC-1 cells and immunized with pgD-E7E6E5 controlled tumor development even after 3 days of tumor cell challenge. In addition, coadministration of pgD-E7E6E5 with DNA vectors encoding pGM-CSF or interleukin-12 (IL-12) enhanced the therapeutic antitumor effects for all mice challenged with TC-1 cells. In conclusion, the present results expand our previous knowledge on the immune modulation properties of the pgD-E7E6E5 vector and demonstrate, for the first time, the strong antitumor effects of the DNA vaccine, raising promising perspectives regarding the development of immunotherapeutic reagents for the control of HPV-16-associated tumors.

Download Full-text

Studies on in Vivo Induction of Cytotoxic T Lymphocyte Responses by Synthetic Peptides from E6 and E7 Oncoproteins of Human Papillomavirus Type 16

Viral Immunology ◽

10.1089/vim.1995.8.165 ◽

1995 ◽

Vol 8 (3) ◽

pp. 165-174 ◽

Cited By ~ 19

Author(s):

ASIS K. SARKAR ◽

GUILLERMO TORTOLERO-LUNA ◽

PRAMOD N. NEHETE ◽

RALPH B. ARLINGHAUS ◽

MICHELE FOLLEN MITCHELL ◽

...

Keyword(s):

Human Papillomavirus ◽

T Lymphocyte ◽

Synthetic Peptides ◽

Cytotoxic T Lymphocyte ◽

Human Papillomavirus Type 16 ◽

E6 And E7 Oncoproteins ◽

E6 And E7 ◽

Lymphocyte Responses ◽

In Vivo Induction

Download Full-text

Restoration of telomeres in human papillomavirus-immortalized human anogenital epithelial cells.

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.961 ◽

1994 ◽

Vol 14 (2) ◽

pp. 961-969 ◽

Cited By ~ 109

Author(s):

A J Klingelhutz ◽

S A Barber ◽

P P Smith ◽

K Dyer ◽

J K McDougall

Keyword(s):

Human Papillomavirus ◽

Epithelial Cells ◽

Telomere Length ◽

Open Reading Frames ◽

Population Doubling ◽

Telomere Shortening ◽

Human Papillomavirus Type 16 ◽

Immortalized Cells ◽

E6 And E7

Loss of telomeres has been hypothesized to be important in cellular senescence and may play a role in carcinogenesis. In this study, we have measured telomere length in association with the immortalization and transformation of human cervical and foreskin epithelial cells by the human papillomavirus type 16 or 18 E6 and E7 open reading frames. By using a telomeric TTAGGG repeat probe, it was shown that the telomeres of precrisis normal and E6-, E7-, and E6/E7-expressing cells gradually shortened with passaging (30 to 100 bp per population doubling). Cells that expressed both E6 and E7 went through a crisis period and gave rise to immortalized lines. In contrast to precrisis cells, E6/E7-immortalized cells generally showed an increase in telomere length as they were passaged in culture, with some later passage lines having telomeres that were similar to or longer than the earliest-passage precrisis cells examined. No consistent association could be made between telomere length and tumorigenicity of cells in nude mice. However, of the three cell lines that grew in vivo, two had long telomeres, thus arguing against the hypothesis that cancer cells favor shortened telomeres. Our results indicate that arrest of telomere shortening may be important in human papillomavirus-associated immortalization and that restoration of telomere length may be advantageous to cells with regard to their ability to proliferate.

Download Full-text